Water-soluble combination products of gossypol and amino acids



Patented Jan. 22, 1952 3- UNITED STATES PATENT OFFICE WATER-SOLUBLECOMBINATION PRODUCTS OF GOSSYPOL AND AMINO ACIDS Aaron M. Altschul and.Leah E. Castillon, New Orleans, La., assignors to the United States ofAmerica, as represented 'by the Secretary of Agriculture No Drawing.Applic ation May 18, 1951,

Serial No. 227,095

8 Claims.

(Granted under the act of March 3, 1883, as

The invention herein described may be manufactured and used by or forthe Government of the United States of America for governmental purposesthroughout the world, without the payment to us of any royalty thereon.

This invention relates to the preparation of non-toxic water-solubleproducts of gossypol by combination with materials of natural origin.

This application is copending with our applications Ser. Nos. 173,220,filed July 11, 1950, issued as Patent No. 2,563,808, and 173,221 filedJuly ll, 1950, and is a continuation in part of our application 173,2l9filed July 11, 1950, now abandoned.

Gossypol is a naturally occurring pigment of cottonseed which is presentto the extent of 0.5 to 1.5 percent by weight of the kernels. Thispigment is concentrated in the pigment glands of the cottonseed andcomprises 35 to 50 percent of the weight of these glands. Certainsolvents such as petroleum naphthas or chlorinated hydrocarbons can beused to extract the oil from flaked cottonseed kernels withoutmaterially affecting the pigment glands or removing substantialquantities of their contents. Other solvents such as alcohols, ethers,and ketones rupture the pigment glands and extract their contents withthe oil present in the seed.

It has been found that oil-free cottonseed meals containing intactpigment glands are toxic when fed in certain specific amounts to certainanimals such as chicks and pigs. When, however, the pigment glandcontents are removed along with the oil, the resulting meal gives noevidence of any physiological toxic efiects. It has been establishedthat intact pigment glands of cottonseed are toxic to animals and.considerable experimental evidence suggests that one of the principalfactors contributing to the toxicity of pigment glands is the gossypolcontained therein.

In the normal practice of processing cottonseed for oil and meal by thehydraulicor continuous screw-press methods, in which heat and pressureare applied to flaked or rolled cottonseed meats, the pigment glands areruptured and their contents react with the surrounding extraglandularmaterials. Whereas in raw cottonseed meats, the amount of free gossypol,that is gossypol which is readily extractable by aqueous acetone orchlorofrom, varies from 0.5 to 1.5 percent, very little free gossypolcan be detected in hydraulic or screw-press meal. .In some of the newlydeveloped methods of processing by solvent extraction of the oil, thecottonseed meats are cooked prior to extraction inorder to facilitateextraction and the oil-free flakes are cooked after amended April 30,1928; 370 O. G. 757) extraction to detoxify them. Gossypol is alsodestroyed by these processes and generally cannot be detected in theoil-free meals.

If, however, the oil is extracted without application of heat bysolvents which do not cause the rupture of the pigment glands or by thenew method of fractionation whereby the pigment glands are separatedintact from the oil and meal (application of Boatner, Hall, andMerrifield, Serial No. 675,118, filed June 7, 1946, now Patent No.2,482,141), the gossypol remains unchanged. By use of the solventextraction method, the gossypol is found in the oil-free meal and in thegland fractionation process, in the separated pigment glands. In eithercase gossypol can be recovered from the meal or pigment glands byextraction with a suitable solvent such as aqueous acetone and can bepurified to yield pure crystalline material.

Gossypol is a new by-product of processing cottonseed and can materiallyincrease the value of cottonseed to the farmer and processor. It hasbeen suggested that gossypol may have pharmaceutical uses and may beused as an insecticide. Biological research with this material and itsapplication to new uses is difficult, however, because of itsinsolubility in water.

We have found that gossypol may be combined with amino acids to form newcombination products which are soluble in water or in basic bufferedsolutions to the extent of approximately 10 percent. These newcombination products have been found to exhibit unobvious and valuablephysiological properties. For example, while a mixture of gossypol andwater is not toxic to goldfish; a dispersion of the gossypol-amino acidcombination products when put into Water in a concentration of one partto 20,000 parts of water kills the fish in two hours. However, while thegossypol-amino acid combination products are toxic to fish, they arenon-toxic to animals. Test rats or mice per cent of which were killedwithin 18 hours by about 0.005 grams-per gram of their weight of puregossypol were unaffected by over twice that amount of a gossypol-glycinecombination product. The non-toxic (to animals) gossypol-amino acidcombination products have been found to cause an increase in the bloodclotting time of animals such as rabbits. Their physiological propertiessuch as toxicity toward fish and not toward animals is even moresurprising in view of the fact that a freshly-prepared gossypol-dextrosecombination product which is also toxic toward fish and also increasesthe blood clotting time of rabbits, is toxic to animals. It is 3 abouttwice as toxic to rats or mice as is pure gossypol.

While the structure of the gossypol-amino acid combination products hasnot been established, it is evident from their properties that theydiffer markedly from the known amine combination products such asdianiline gossypol which are characterized by their water insolubilityand physiological inactivity. Similarly, since aqueous solutioncontaining an aqueous extract of a commercially prepared screw-pressedcottonseed meal which had been subjected to the conditions of heat andmoisture known to produce what has been referred to as bound gossypol,exhibited no harmful effects upon fish (when the amount of mealextracted contained an amount of gossypol equivalent to that shown toyield a similar extract which is toxic to fish when the gossypol waspresent in the form of a gossypol-amino acid combination product) it isevident that the gossypol-amino acid combination products are diiferentsubstances from such known gossypol complexes.

Substantially any alpha-amino carboxylic acid which is appreciablysoluble in an aqueous solution of a pH of about 9 or above can suitablybe employed in the production of the gossypol-amino acid combinationproducts. Such acids include glycine, leucine, threonine, serine, lysineaspartic acid, glutamic acid, and the like. A particularly suitableclass of alpha-amino carboxylic acids for employment in the productionof the present invention are amino-substituted alkanoic acids containingan alpha-amino group which are soluble in an aqueous solution of pH 10or more. It has been demonstrated that either the neutral or basicmembers of this class of amino acids produce the combination productshaving the unique physiological properties mentioned above.

The production of the combination products is conducted in a solvent forboth the gossypol and the amino acid. A basic aqueous solution,preferably an alkali metal hydroxide solution, having a pH of at least10 is a particularly suitable solvent. While gossypol (free or dissolvedin a solvent) is known to combine with some solid substances at or aboveroom temperature, such combination products (for example, boundgossypol) exhibit substantially none of the unique and valuablephysiological properties of the gossypolamino acid combination products.

When dissolved in an aqueous alkaline solution containing an amino acid,gossypol reacts rapidly at room temperature, and while temperatures offrom C. to the decomposition temperature of the reactants can suitablybe used, the reaction is preferably conducted at about normal roomtemperature. The reaction is rapid enough at room temperature to beproductive of good yield when the solution is neutralized with aninorganic acid substantially immediately after dissolving orsolubilizing the gossypol. The reaction can also be conducted bydissolving the gossypol and adding the amino acid to the resultingsolution.

The combination products can be isolated by removing the solvent afterneutralization to a pH of from 7 to 7.2 at any temperature below thedecomposition temperature. However, their isolation by the removal ofthe water by sublimation at 0 C. or below is preferred. They can also beisolated by removing by filtration, centrifugation, extraction, and thelike methods, the ine soluble materials formed when the pH of thesolution is lowered to from about 4.5 to 7. The remaining solvent canthen be removed by evaporation, preferably at a temperature of not morethan 0 C.

The process of the invention can be conducted in a continuous orbatch-wise manner, but the latter mode of operation is preferred.

The proportions of gossypol and amino acid can be varied widely, ingeneral, from about 1:10 to 10:1. We have demonstrated that gossypolcombination products exhibiting the above unique properties are formedwhen gossypol and glycine are combined in ratios of from 9:1 to 129parts.

The products of our invention and the methods for preparing theseproducts are further illustrated but not limited by the followingexamples.

Example 1.Three grams of glycine was added to 250 ml. distilled water,and three grams of pure gossypol was added to this glycine solution.

Aqueous alkali was added while the mixture was being stirred until thegossypol was solubilized, and the pH of the solution was approximately10.3. Hydrochloric acid solution was added dropwise to bring the pH ofthe solution to 7.0. The solution was then frozen, and then,

lyophilized. This material is soluble in water at neutral pH and toxicto goldfish in dilutions of 1 part to 20,000 parts of water.

Example 2.One gram of l(+) lysine-monohydrochloride was added to 200 ml.of distilled water. While the solution was being stirred, one gram ofgossypol and a sufficient quantity of aqueous sodium hydroxide was addedto dissolve the gossypol. When complete solution was achieved,hydrochloric acid solution was added until the pH was lowered to 7.0,the solution frozen, and dried by lyophilization. A yellow, brittleresidue, weighing 1.87 grams was obtained. This material is soluble inwater at neutral pH and toxic to goldfish in dilutions of 1 part to20,000 parts of water. However, this material was not toxic to mice whengiven at a dosage level of 12 grams of gossypol-lysine per kilogram bodyweight of mice. Much smaller doses of gossypol or pigment glands werelethal to the test animals within 24 hours.

Example 3.-Twenty-five grams of ammoniafree glycine was dissolved in 600milliliters of distilled water. Twenty-five grams of pure gossypol wasadded to this solution. Dilute aqueous sodium hydroxide was addeddropwise until the pH of the solution was 11.0. The gossypol wassolubilized at this pH. Immediately thereafter, an aqueous solution ofconcentrated hydrochloric acid was added dropwise until the pH of thesolution was reduced to 7.0 to 7.2. During the course of thispreparation, the solution was constantly agitated. The solution was thenfrozen and lyophilized, i. e., the water was removed from the materialin a frozen state under conditions of high vacuum, low pressure and lowtemperature within 48 hours. A yield of 50 grams of fluffy, light yellowproduct was obtained. The dry product was soluble in water at neutralpH.

This product was not toxic when administered orally to mice or rats, inaqueous media, as large, acute doses. At a dosage level of 12.0 grams ofthis product per kilogram body weight of the test animals, this productproduced no visible effects. On the other hand, pure gossypol had anEDso value (the dose lethal within 48 hours to 50 percent of the testanimals) of 4.8 grams :0.6 gram per kilogram body weight of mice. TheEDso value for separated pigment glands was 2.3 grams :011 gram perkilogram body weight. A sample of freshly-prepared water-solublegossypol-dextrose combination product (described and claimed in ourcopending application, Serial No. 173,220, filed July 11, 1950) wasabout as toxic for mice as the pure gossypol, and had an EDso value of2.6 grams 10.17 gram per kilogram body weight. The EDsc values werecalculated according to the method of Miller and Tainter.

Samples of the gossypol-glycine combination product were stored for fivemonths at l8 C. ,1

and 28 C. During this storage period, the samples remained non-toxic andwater-soluble.

Example 4.--Fortyfive grams of pure gossypol was added to five grams ofglycine dissolved in 500 milliliters of distilled water. Water was addeduntil the volume of the solution was approximately one liter. The pH ofthe solution was adjusted to 11.0 by the addition of aqueous sodiumhydroxide. The gossypol was solubilized at this pH. While the solutionwas constantly agitated, aqueous hydrochloric acid solution was addeddropwise until the pH of the solution was lowered to 7.0 to 7.2. Thesolution was then frozen and lyophilized. The dried, yellow product wassoluble in water at neutral pH.

This product was not toxic when given orally in large, acute dosages torats. However, 3.0 grams of pure gossypol per kilogram of body weight ofthe rats, or 0.9 to 1.3 grams of pigment glands per kilogram body weightkilled 50 percent of the test animals within 24: hours.

Example 5.--Forty-five grams of glycine was dissolved in one liter ofdistilled water. Five grams of pure gossypol was added to this solution.

Then aqueous sodium hydroxide solution was added until the pH of thesolution was increased to 11.0. While the solution was being stirred,aqueous hydrochloric acid solution was added until the pH was lowered to7.0 to 7.2. The solution was then frozen and lyophilized. The driedproduct was soluble in water at neutral pH, and was not toxic whenadministered orally in acute, large dosages to rats. Much smaller Havingthus described our invention, what we claim is:

1. A water soluble gossypol-amino acid combination product, prepared byreacting gossypol and an amino acid in an alkaline aqueous mediuni; andcharacterized by toxicity to goldfish and non-toxicity to rats or mice,the amino acid being an alpha-amino alkanoic carboxylic acid which issoluble in an aqueous solution of an alkali metal hydroxide having a pHof at least 9.

2. The product of claim 1 where the amino acid is glycine.

3. The product of claim 1 where the amino acid is lysine.

4. A process for the production of a gossypolamino acid combinationproduct, which process comprises: reacting in an alkaline aqueous media,gossypol and an amino acid; neutralizing the media with an inorganicacid; and removing the water at a temperature of below about 20 C.

5. A process for the production of a gossypolamino acid combinationproduct, which process comprises: dissolving an amino acid in water;dissolving gossypol in the same solution by raisin the pH of thesolution to at least 10 by the addition of an alkali metal hydroxide;neutralizing the solution by addition of an inorganic acid; cooling thesolution to below 0 C.; and removing the water by sublimation.

6. A process for the production of a gossypolamino acid combinationproduct, comprising, reaching gossypol and an amino acid in an alkalineaqueous medium.

7. A water soluble gossypol-amino acid combination product, the aminoacid being an alphaamino alkanoic carboxylic acid of the classcorresponding in structure to naturally occurring aliphatic amino acids.

8. The product of claim 1 in which the amino acid is a naturallyoccurring amino acid.

AARON M. ALTSCHUL. LEAH E. CAS'IILLON.

REFERENCES CITED The following references are of record in the file ofthis patent:

Gallup: J. Biol. Chem. 16, pp. 43-53 (1928).

1. A WATER SOLUBLE GOSSYPOL-AMINO ACID COMBINATION PRODUCT, PREPARED BYREACTING GOSSYPOL AND AN AMINO ACID IN AN ALKALINE AQUEOUS MEDIUM; ANDCHARACTERIZED BY TOXICITY TO GOLDFISH AND NON-TOXICITY TO RATS OR MICE,THE AMINO ACID BEING AN ALPHA-AMINO ALKANOIC CARBOXYLIC ACID WHICH ISSOLUBLE IN AN AQUEOUS SOLUTION OF AN ALKALI METAL HYDROXIDE HAVING A PHOF AT LEAST 9.